Project Summary: Our long-term goals are to understand the catalytic mechanism and biological specificity of virus-encoded enzymes that modify RNA ends. We are studying viral mRNA capping and viral tRNA repair as paradigms of RNA transactions that enable virus replication. The studies of capping provide fresh insights to the evolution of a uniquely eukaryotic mRNA processing event and are opening up new approaches to antiviral drug discovery targeted at viral mRNA cap formation. The current research plan is focused on the RNA triphosphatase and RNA (guanine-N7) methyltransferase components of the poxvirus mRNA capping apparatus and includes specific aims to: (a) interrogate whether the viral capping activities are essential for poxvirus replication in cell culture;(b) map the active site of the cap methyltransferase and determine its structure with substrates and inhibitors bound;(c) evaluate a sinefungin-based transition state analog as a specific inhibitor of poxvirus cap methylation;(d) determine the structure of the viral RNA triphosphatase and identify novel inhibitors via high-throughput screening. Our studies of tRNA repair are illuminating the evolutionary transitions from RNA-world to DNA-world enzymology. This proposal focuses on bacteriophage T4 Pnkp, a bifunctional 5'kinase/3'phosphatase that heals broken tRNA ends. Pnkp functions in vivo to antagonize a host antiviral response that blocks viral protein synthesis through tRNA depletion. Our aim is to elucidate the mechanism of the 3'-phosphatase domain by capturing structures of the enzyme at different steps along the reaction pathway. Relevance: Exploitation of new molecular targets for treatment of poxvirus infections is a pressing issue, given the concerns that smallpox could be used as a bioterror weapon and the risk of complications of vaccinia infections if a prophylactic vaccination program is resumed. The outbreak of human monkeypox infections in the US in 2003 highlighted the risks of re-emergence of human poxvirus disease. mRNA capping enzyme is an attractive therapeutic target for smallpox because the active site and catalytic mechanism of the poxvirus RNA triphosphatase are completely different from that of the human RNA triphosphatase. Poxvirus cap methyltransferase is also distinguished from the human counterpart by its reliance on a unique virus-encoded regulatory subunit.